Peripheral inflammatory response in people after acute ischaemic stroke and isolated spontaneous cervical artery dissection

The systemic inflammatory response following acute ischaemic stroke remains incompletely understood. We characterised the circulating inflammatory profile in 173 acute ischaemic stroke patients by measuring 65 cytokines and chemokines in plasma. Participants were grouped based on their inflammatory response, determined by high-sensitivity C-reactive protein levels in the acute phase. We compared stroke patients’ profiles with 42 people experiencing spontaneous cervical artery dissection without stroke. Furthermore, variations in cytokine levels among stroke aetiologies were analysed. Follow-up samples were collected in a subgroup of ischaemic stroke patients at three and twelve months. Ischaemic stroke patients had elevated plasma levels of HGF and SDF-1α, and lower IL-4 levels, compared to spontaneous cervical artery dissection patients without stroke. Aetiology-subgroup analysis revealed reduced levels of nine cytokines/chemokines (HGF, SDF-1α, IL-2R, CD30, TNF-RII, IL-16, MIF, APRIL, SCF), and elevated levels of IL-4 and MIP-1β, in spontaneous cervical artery dissection (with or without ischaemic stroke as levels were comparable between both groups) compared to other aetiologies. The majority of cytokine/chemokine levels remained stable across the study period. Our research indicates that stroke due to large artery atherosclerosis, cardioembolism, and small vessel occlusion triggers a stronger inflammatory response than spontaneous cervical artery dissection.

Certain factors, such as the severity of the stroke and the patient's age which influence the high disability and mortality rates associated with stroke 1 , remain beyond our control 2 .However, other factors such as inflammation related to stroke, have been explored as new targets to prevent stroke-related complications and improve functional outcomes 3 .These concepts were adopted with the emergence of novel therapeutic targets aimed at reducing peripheral inflammation in individuals after myocardial infarction, since elevated cytokine levels have been linked to unfavourable clinical outcomes in ischaemic stroke patients 4 .Long-term studies have revealed systemic inflammation that emerges not only within minutes to hours after acute ischaemic stroke (AIS), but persists for several days and drives complications 5,6 .Although several receptor antagonists have been studied for their potential to reduce circulating inflammatory cytokine levels and improve clinical outcomes after AIS, their effectiveness remains uncertain.Large ongoing trials 7 and conflicting results from smaller studies 8,9 , reflect our incomplete understanding of immune processes following cerebral ischaemia.Nevertheless, some ongoing trials show promise, such as the investigation of colchicine to prevent inflammation 10 .C-reactive protein (CRP), a prominent acute-phase protein, is one of the most extensively studied blood markers for inflammation and is implemented in many laboratory panels used in people with AIS 11 .High CRP levels have been found not only to be associated with an increased risk of developing ischaemic stroke 12 , but also to predict functional outcome and mortality when measured at the time of admission [13][14][15][16][17][18] .Other prominent inflammatory markers in stroke research include interleukin (IL)-6, IL-1, IL-10, tumour necrosis factor (TNF)-α and TNF-β, as well as the chemokines monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and chemokine ligand-5 [19][20][21] .Most of these cytokines and chemokines can be found upstream of CRP, but their effect on functional outcome is still not as sufficiently studied as the effect of CRP.Thus, more research is needed to clarify the role of these cytokines and chemokines in cerebrovascular disease, including their association with stroke severity, stroke aetiology, and functional outcomes.Also the precise impact of the post-ischaemic inflammatory response on brain recovery, especially in advanced stages, remains incompletely understood 6,22 .
In recent years, various smaller panels of pro-and anti-inflammatory cytokines and chemokines have been studied in patients with stroke.The aim of this study was to measure plasma levels of 65 different cytokines, chemokines, soluble surface molecules, and immune receptors using one methodology in patients with AIS to better understand the interplay between pro-and anti-inflammatory cytokines in the acute phase.Furthermore, we aimed to evaluate possible differences between patients showing a higher peripheral inflammatory response in the acute phase, as indicated by high CRP levels in the blood, and patients with normal CRP levels.We also investigated if levels of these 65 cytokines and chemokines remain stable in follow-up samples up to twelve months and examined their predictive value for functional outcome.Finally, we performed subtype-stratified analyses as only a few studies so far have assessed potential differences among various AIS aetiologies.

Classification of stroke severity and parameters of functional outcome after twelve months
For acute assessment of stroke severity, the National Institutes of Health Stroke Scale (NIHSS) was evaluated upon admission, along with the modified Rankin Scale (mRS) score.People were grouped based on their NIHSS scores into minor (0-5), moderate (6-15), or severe (≥ 16) stroke categories 29 .
Long-term functional outcomes based on the mRS were also assessed at the 12-month follow-up visit, during which plasma samples were taken.Missing mRS follow-up data in AIS patients without this 12-month followup visit was completed through retrospective chart review of all available electronic medical files.The collected mRS-follow-up data is addressed to as "mRS at last clinical follow-up".Good functional outcome was defined as mRS ≤ 2.

Statistical analysis
Statistical analyses were performed using GraphPad Prism-10 (GraphPad Software Inc., La Jolla, California, United States) and SPSS-27 (IBM SPSS Statistics, Armonk, NY).All figures were created by the authors using GraphPad Prism-10.
The primary aim of the study was to compare the peripheral cytokine/chemokine profile in people after AIS classified by high-sensitivity CRP levels at admission, those AIS patients with unaltered peripheral inflammatory responses at admission, and people without stroke having sCeAD with local symptoms only.For overall multiple group comparisons, the non-parametric Kruskal-Wallis test was used.P-values were corrected for multiple (n = 65) comparisons using Bonferroni's correction.Effect size coefficients η 2 were calculated from the Kruskal-Wallis statistics using the formula η2 = (H−k+1) (n−k) (H = Kruskal-Wallis H value; k = number of groups; n = number of total observations) and classified as either small (0.01 to < 0.06), moderate (0.06 to < 0.14), or large (η 2 ≥ 0.14) according to Cohen, 1988 30 .Dunn's multiple comparisons tests were used to compare subgroups.The necessary sample size for the Kruskal-Wallis test (for 5% α and 80% power (1-ß)) was calculated using G*Power software based on F values calculated from η 2 values (F = η 2 (1−η 2 ) ).To detect a moderate effect size of F > 0.25 (corresponding to η 2 > 0.06) it was determined to be > 159 (total sample size) and to detect a large effect size of F > 0.40 (corresponding to η 2 > 0.14) it was determined to be > 66 (total sample size).
Secondary aims were (i) to perform subtype-stratified analyses to assess potential differences among various AIS aetiologies (using the non-parametric Kruskal-Wallis test).The necessary sample size was calculated as described above and was determined to be > 180 (total sample size) to detect a moderate effect size of F > 0.25 (corresponding to η 2 > 0.06) or > 76 (total sample size) to detect a large effect size of F > 0.40 (corresponding to η 2 > 0.14).Moreover, a multivariate linear regression model was used to predict the role of clinical (age, sex, ischaemic stroke, aetiology, NIHSS, and mRS score) and laboratory (CRP) parameters on baseline plasma levels of significantly altered analytes.Cytokine levels were log10 transformed to meet the assumption of the model, and results are shown as estimates b (with standard error of mean) and standardised estimates ß (with 95% confidence intervals).
(ii) To analyse the associations of altered cytokines/chemokines with demographic, clinical, and laboratory characteristics of the acute event (using non-parametric Spearman's rank correlation test, Mann-Whitney U test, chi-square test, or Fisher's exact test).Due to limited statistical power and the large number of statistical comparisons, we only show effect sizes, but no p-values.Effect size coefficients R were calculated from the Mann-Whitney U statistics using to the formula R = z √ N (z = Mann-Whitney U z-value; N = total number of ranks).Calculated R and Spearman ρ effect sizes were classified as either small (-0.3 to -0.1, or 0.1 to 0.3), moderate (-0.5 to -0.3, or 0.3 to 0.5), or large (< -0.5 or > 0.5) according to Cohen, 1988 30 .Effect sizes were then grouped by k-means clustering (Pearson's correlation) and are shown as a heat map.
(iii) To investigate the association of baseline cytokine/chemokine levels for functional outcome (by multivariate binary logistic regression analysis).
(iv) To examine if levels of cytokines and chemokines remain stable in follow-up samples in a subgroup of AIS patients up to twelve months and to analyse their association with AIS subtypes and outcome (using the non-parametric Friedman test or Kruskal-Wallis test with Dunn's multiple comparison tests).
Statistical significance was defined as two-sided p-value < 0.05 after correction for multiple comparisons using Bonferroni's correction.

Ethics approval and consent to participate
The use of frozen samples from a biobank for this study was approved by the local ethics committees of the Medical University Innsbruck, Austria (ReSect study: EK#UN5072, 325/4.1,29.03.2019 31 ; STROKE-CARD study: EK# UN2013-0045 and is registered with Clinical-Trials.govnumber NCT02156778 (03.01.2014) 23 ).All patients or their legal representatives gave written informed consent to diagnostic procedures and biological sample storage for research purposes.

Baseline characteristics of study participants
Baseline characteristics of patients after AIS classified according to their baseline CRP levels and characteristics of sCeAD-nonAIS patients are presented in Table 1.Among all AIS patients, 49 (28.3%) were classified as having baseline CRP levels of > 5 mg/l, and 124 (71.7%) had CRP levels ≤ 5 mg/l.Cerebrovascular risk factors of all stroke patients grouped according to the underlying aetiology are listed in Table 2, along with Table S1 of the Supplementary file 1.
In contrast, MIP-1β and IL-4 showed a completely different picture with a positive association with sCeAD, CRP, and many coagulation parameters.Furthermore, especially MIP-1β showed a negative association with age, CE, LAA, SVO, INR, and hypertension among many more.www.nature.com/scientificreports/IL-2R, CD30, TNF-RII, IL-16, MIF, and APRIL were higher in AIS patients with mRS 3-5, in the multivariate model, only higher age and higher NIHSS assessed in the acute phase were associated with mRS > 2 (Supplementary Table S7).

Stability of plasma cytokine/chemokine levels after month three and month twelve of follow-up in a subgroup of AIS patients
Plasma cytokine/chemokine levels were measured in a subgroup of people with AIS at a median of 3.3 (range 2.3-4.3)months, and for the last follow-up measurement at a median of 12.6 (range 11.1-14.2) months after the acute event.This subgroup included 30 patients with stroke due to LAA, 34 with CE stroke, and 15 with SVO stroke.Clinical and laboratory characteristics at these follow-up visits are shown in Supplementary Table S8 and Fig. S2.
A decrease in plasma levels comparing baseline and both follow-up time points was observed for HGF, and these plasma levels remained stable between three-month and twelve-month follow-up time points.Similarly, TNF-RII levels decreased three months after the acute event, while plasma CD30 levels increased during followup.Statistical testing to assess differences between stroke aetiology groups regarding baseline and follow-up cytokine/chemokine levels was not performed due to the limited sample size.

Discussion
Our investigation into the systemic inflammatory response following AIS revealed distinct plasma concentrations of HGF, SDF-1α, and IL-4 compared to individuals with sCeAD-nonAIS.Our study highlighted unique inflammatory profiles between sCeAD and other stroke aetiologies, though no differences were found between sCeAD with or without stroke.The comparison of all 65 cytokines and chemokines among the four aetiology groups (sCeAD, LAA, CE, and SVO) revealed differences for eleven analytes.Plasma levels of HGF, SDF-1α, IL-2R, CD30, TNF-RII, IL-16, MIF, APRIL, and SCF were lower in sCeAD than in LAA, CE or SVO, but IL-4 and MIP-1β levels were higher in sCeAD.Elevated CRP levels were not associated with these cytokines and chemokines.In summary, our results indicate substantial differences in the inflammatory response between dissection (with or without stroke) and other stroke aetiologies (LAA, CE, and SVO).
Many of the proteins showing differences in our study play established roles in stroke or cardiovascular diseases.HGF, for instance, is recognised as an endothelium-specific growth factor with diverse beneficial functions, including anti-inflammatory, anti-fibrotic, and pro-angiogenetic properties 32 .It is primarily released in response to endothelial injury, leading to elevated levels observed in cardiovascular conditions such as atherosclerosis 33 , acute myocardial infarction 34 , or AIS 35 .Previous research has linked higher HGF levels with poor outcomes in these diseases 36,37 , likely due to increased endothelial release.Interestingly, our study revealed a decrease in HGF levels three months after the acute stroke event, suggesting its involvement not only in the inflammatory response to stroke but also in reflecting the response to cerebral ischaemia itself during the acute phase.In contrast to HGF, levels of most other cytokines/chemokines remained elevated up to one year, indicating a more generalised inflammatory response, as ischaemia itself would not be expected to drive inflammatory cytokine and chemokine levels to a similar extent after twelve months.This is consistent with previous findings where upregulated proteins during the acute phase of stroke remained elevated in follow-up measurements 38 .
SDF-1α is produced by activated astrocytes, microglia, and vascular endothelial cells.It plays a crucial role in recruiting monocytes locally to the injured brain region after ischaemic stroke [39][40][41] and mobilises endothelial progenitor cells, thus promoting angiogenesis 42 .Elevated SDF-1α levels have been observed in individuals with cardiovascular risk factors 43,44 , and plasma levels have been linked to adverse cardiovascular outcomes in people with coronary artery disease 45 .Similarly, elevated levels of IL-2R have been reported in coronary artery atherosclerosis 46 and associated with various cardiovascular risk factors, including diabetes mellitus and hypertension 47 , as confirmed in our study.
Members of the TNF receptor superfamily such as CD30, TNF-RII, and APRIL, expressed on immune cells 48,49 were found to be upregulated in the blood of ischaemic stroke patients in the sub-acute phase [50][51][52] .APRIL was shown to protect against atherosclerosis by binding to heparin-sulphate proteoglycans in mice, and decreased serum levels are associated with long-term cardiovascular mortality in individuals with atherosclerosis 53 .IL-16, released by activated CD8+ T cells, contributes to the inflammatory response following ischaemic stroke.It activates CD4+ T cells, monocytes, macrophages, and dendritic cells, leading to the expression of various inflammatory cytokines.This process drives secondary brain damage 19,[54][55][56] .MIF, a pleiotropic inflammatory mediator with chemokine-like functions, promotes leukocyte migration to inflammatory sites, including atherosclerosis 57 .MIF is not only produced by monocytes, macrophages, B-or T-cells, but also by endothelial and epithelial cells 58 .
SCF, produced by bone marrow stromal cells, induces neuroproliferation, reduces infarct size, and improves functional outcome in ischaemic stroke models 59,60 .
Distinct findings of higher plasma IL-4 and MIP-1β levels were observed in sCeAD patients compared to other stroke aetiologies.IL-4 has an anti-inflammatory role and is a critical regulator of M2 polarization.Following its secretion by neurons in response to ischaemia, it stimulates microglial phagocytosis and enables clearance of apoptotic neurons.Consequently, it promotes long-term recovery of microglia/macrophages 61 .MIP-1β, a chemokine secreted by various vascular and hematopoietic cells 62 , is linked to atherosclerosis 63 .Elevated blood levels of MIP-1β are reported in people with this condition 64 .However, such elevations were not observed in cohorts of stroke patients 21 , warranting additional investigation into underlying mechanisms regarding its differential expression in distinct cardiovascular pathologies.IL-4, along with MIP-1β, was associated with distinct clinical parameters, showing mostly positive Spearman's correlation values with platelet count, PT, AT-III, or CRP in our study.Interleukins play a unique role in megakaryopoiesis, thrombopoiesis, and platelet function, with the majority having positive effects.However, a subset such as IL-4 or IL-1α has been reported to possess inhibitory properties on megakaryocyte differentiation, thereby inhibiting platelet production [65][66][67][68] .This contradicts the findings of our study and warrants further investigation.This includes a more comprehensive characterization of the study participants, particularly regarding haematological disorders that may have influenced these outcomes.
Experimental studies and clinical trials using above-described cytokines and chemokines have shown promising results in ischaemia treatment.Preclinical studies on rats or mice demonstrated the positive effect of HGF on protecting blood-brain barrier integrity, reducing infarct volume, and improving functional recovery after stroke [69][70][71][72] .Clinical trials testing HGF in peripheral or coronary artery ischaemia proved good tolerance [73][74][75] .However, to our knowledge, such trials for ischaemic stroke have not been conducted yet.Other therapeutic approaches involving cytokines and chemokines have also yielded promising results, especially in animal models [76][77][78][79] .
In our study cohort, individuals after sCeAD, whether experiencing a stroke or only local symptoms, had the lowest levels of cytokines compared to other stroke aetiology groups during the acute phase.This is an important finding, as it distinguishes our study from previous research.While another research group examined a similar panel of cytokines and chemokines in the blood of AIS patients, they neither conducted a comparative analysis across different AIS aetiology groups nor included such a substantial cohort of patients experiencing AIS due to sCeAD, including those presenting solely with local symptoms 21 .However, another study, albeit with a smaller sample size of 29 patients diagnosed with sCeAD leading to AIS, reported comparable findings of decreased cytokine levels in this aetiology of AIS compared to other causes 38 .
Our results also indicate that only higher age and higher NIHSS score, but not levels of any cytokine/ chemokine, were associated with mRS > 2 after twelve months, indicating unfavourable outcomes.
There are several limitations to this study.Firstly, it is retrospective and includes a relatively small number of AIS patients.Although the sample size was sufficient to detect a moderate effect size for the primary aim, unequal group sizes based on CRP levels and TOAST aetiologies limit the statistical power.To address this, we used Bonferroni's correction for multiple comparisons and focused on cytokines and chemokines with large effect sizes.However, the small sample size for most subgroup analyses, especially when classifying AIS patients by mRS scores (≤ 2 or > 2), further limits statistical power.We hypothesize that more cytokine/chemokine levels might differ between groups, particularly when comparing stroke due to sCeAD and other aetiologies.Therefore, larger studies including more AIS patients and individuals with sCeAD are needed to confirm our results and establish causality between cytokine levels and clinical factors.
Secondly, the retrospective design and inclusion of stroke patients from two different trials (STROKE-CARD trial, ReSect-study) resulted in differing clinical data sets and some missing clinical information, potentially introducing reporting bias.The time span between the onset of AIS or sCeAD symptoms and blood sampling for cytokine/chemokine assessment varied and was not standardised, as blood sampling was always performed at 8 am.
Moreover, this study does not accurately reflect the natural distribution of ischaemic stroke causes.In this study, patients with sCeAD-AIS constitute the majority, whereas patients with CE, which typically represents a major aetiology in AIS, are underrepresented.Since sCeAD accounts for approximately 1-2% of stroke patients 80 , its prevalence in this study is disproportionate.This also affects the median age of participants, as sCeAD leading to AIS or local symptoms predominantly occurs in young and middle-aged adults, whereas AIS due to other aetiologies commonly affects older people 81 .The selection of a group of people with sCeAD-nonAIS might impact the altered cytokine profile observed.While some epidemiological observations suggest a link between inflammation and sCeAD, particularly following infections [82][83][84] , the exact underlying pathophysiological mechanisms remain unclear.Reported infections include respiratory and urinary tract infections, and gastroenteritis, typically appearing within four weeks before the sCeAD event 85 .Notably, these infections often present with mild symptoms and usually resolve before hospitalization 86 , so we speculate they may not influence the cytokine/ chemokine profile observed post-sCeAD event in our study.
We categorised participants into CRP groups of ≤ 5 mg/l or > 5 mg/l.It is crucial to acknowledge that CRP, as an acute-phase protein, is a nonspecific indicator of inflammation.Elevated CRP levels can result from infections 87 , tissue damage associated with atherosclerosis or ischaemic stroke 88 , chronic diseases 89 , or autoimmune disorders 90,91 .Therefore, there is a possibility of misclassifying AIS patient into the > 5 mg/l group if elevated CRP levels did not originate from the acute stroke event.
Finally, we evaluated long-term functional outcomes at twelve months and at the last clinical follow-up visit, with a median of 5.1 years post-stroke in the > 5 mg/l CRP group and 1.4 years in the ≤ 5 mg/l CRP group using the mRS score 92 .Unfortunately, neuroimaging scans were not included to investigate potential associations between infarct volume and baseline cytokine/chemokine levels.Ischaemic lesion volume has been suggested as a surrogate imaging biomarker in the early clinical phase of AIS to predict functional outcomes 93 .Previous studies have established an independent relationship between ischaemic lesion volume and functional outcome 94 , with a strong correlation between ischaemic stroke infarct volume on follow-up CT and MRI scans and the mRS score 95 .However, these findings are influenced by factors such as ischaemic lesion location and the timing of lesion volume measurement or mRS assessment 96 .Furthermore, non-modifiable factors like older age, stroke severity, or pre-stroke dependency, as well as complications during hospitalization, including pneumonia, increased intracranial pressure, or cerebral oedema, affect outcomes after three months 97,98 .Unfortunately, we did not assess early in-hospital complications in our study cohort.

Table 1 .
Demographics, clinical and routine blood parameters of people after acute ischaemic stroke and cervical artery dissection with local symptoms only.Values are expressed as percentages or median (min- max).AIS acute ischaemic stroke, AT-III antithrombin-III, CE cardioembolism, CRP C-reactive protein, HbA1c glycated haemoglobin (A1c), HDL-C high-density lipoprotein cholesterol, HGF hepatocyte-growthfactor, INR international normalised ratio, IL interleukin, LAA large artery atherosclerosis, LDL-C low-density lipoprotein cholesterol, mRS modified Rankin Scale, NIHSS National Institutes of Health Stroke Scale, PT prothrombin time, sCeAD spontaneous cervical artery dissection, sCeAD-nonAIS spontaneous cervical artery dissection with local symptoms only (i.e.no ischaemic stroke), SDF-1α stromal-cell-derived factor-1α, SVO small vessel occlusion. 1Cytokine levels were only measured at month three and twelve follow-up time point in a subgroup of AIS patients, not at the last clinical follow-up where the mRS was assessed.*p < 0.05 vs sCeAD-nonAIS, † p < 0.05 vs stroke with CRP ≤ 5 mg/l patients.Stroke with CRP > 5 mg/l (n = 49) Stroke with CRP ≤ 5 mg/l (n = 124) sCeAD-nonAIS (n = 42)

Table 2 .
Baseline risk factors of people after ischaemic stroke and cervical artery dissection.Patients with cervical artery dissection leading to stroke (n = 94) and those with local symptoms only (n = 42) were grouped together as no differences between these two were found.Values are expressed as percentages or median (minmax).BMI body mass index, CCA /ICA common carotid artery/internal carotid artery, TIA transient ischaemic attack. 1 Including 94 patients with cervical artery dissection leading to stroke and 42 patients having cervical artery dissection with local symptoms only; *p < 0.05 vs patients with cervical artery dissection using chisquare test or Fisher's exact test.